Bottom Line:
We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs.Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative.The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing.

Affiliation: Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA. jdrake@northnet.org

ABSTRACTB cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II-restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II- containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igalpha subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.

Figure 5: Identification of a putative CIIV-marker protein that is immunologically related to Igα subunit of the BCR. (A) Identification of a 50-kD protein enriched in CIIV-containing FFE fractions. A20μWT cells were fractionated by FFE and the distribution of markers for plasma membrane (PM, unshifted class II), endosomes/lysosomes (E/L, β-Hexosaminidase), and CIIV (anodally shifted class II) were determined. The position and size in kilodaltons, of the molecular mass standards is indicated at the right. The distribution of the Igα subunit of the BCR was determined by Western blot analysis using a rabbit antiserum against Igα. The 32-kD Igα protein (indicated by B) was detected at the highest levels in PM-containing FFE fractions containing and to a lesser extent in fractions containing endosomes–lysosomes and CIIV. Additionally, a 50-kD protein (A) was selectively enriched in the CIIV-containing FFE fractions (fractions 52–59). The 50-kD protein is also detectable in the unfractionated LDM. Illustrated are results representative of five independent experiments. (B) The 50-kD putative CIIV-marker protein is immunologically related to the Igα subunit of the BCR. Preparative Western blots of A20 LDMs were probed with the rabbit anti-Igα antiserum and washed. The antibodies against Igα and the 50-kD protein were affinity purified by excising the regions of the blot containing the anti-Igα and anti-50-kD proteins (and bound antibodies) and then eluting the bound antibodies. The affinity-purified antibodies, as well as uncut antiserum, were then used to probe a Western blot of unfractionated LDM. The uncut serum (lane 1) recognized both the 32-kD Igα (arrow B) as well as the 50-kD protein (arrow A) in the LDM. Although the affinity-purified anti-Igα (lane 2) did recognize Igα, it failed to recognize the 50-kD protein present in the LDM. (In some experiments, the affinity-purified anti-Igα antibodies did demonstrate reactivity towards the 50-kD protein.) Importantly, antibodies affinity purified on the 50-kD protein (lane 3) recognized both the 32-kD Igα protein (arrow B) as well as the 50-kD protein (arrow A), indicating an immunological relationship between these two proteins. (Immunoreactivity of the affinity-purified anti-50-kD antibodies against Igα was observed in every experiment.) Illustrated are results representative of three independent experiments.

Mentions:
To this end, we examined the steady state distribution of the Igα subunit of the BCR. As shown in Fig. 5 A, the majority of the 32-kD Igα protein (arrow B) is present in PM-containing FFE fractions with lesser amounts detected in endosome–lysosome and CIIV-containing fractions. The same distribution was also found for the Igβ subunit of the BCR as well as the heavy and light chain subunits of both the huBCR and muBCR (data not shown), suggesting that the subunit composition of the BCRs in these compartments is similar. Surprisingly, the anti-Igα antiserum also recognized a second, 50-kD protein (Fig. 5 A, arrow A) that appears to be selectively enriched in CIIV-containing FFE fractions.

Figure 5: Identification of a putative CIIV-marker protein that is immunologically related to Igα subunit of the BCR. (A) Identification of a 50-kD protein enriched in CIIV-containing FFE fractions. A20μWT cells were fractionated by FFE and the distribution of markers for plasma membrane (PM, unshifted class II), endosomes/lysosomes (E/L, β-Hexosaminidase), and CIIV (anodally shifted class II) were determined. The position and size in kilodaltons, of the molecular mass standards is indicated at the right. The distribution of the Igα subunit of the BCR was determined by Western blot analysis using a rabbit antiserum against Igα. The 32-kD Igα protein (indicated by B) was detected at the highest levels in PM-containing FFE fractions containing and to a lesser extent in fractions containing endosomes–lysosomes and CIIV. Additionally, a 50-kD protein (A) was selectively enriched in the CIIV-containing FFE fractions (fractions 52–59). The 50-kD protein is also detectable in the unfractionated LDM. Illustrated are results representative of five independent experiments. (B) The 50-kD putative CIIV-marker protein is immunologically related to the Igα subunit of the BCR. Preparative Western blots of A20 LDMs were probed with the rabbit anti-Igα antiserum and washed. The antibodies against Igα and the 50-kD protein were affinity purified by excising the regions of the blot containing the anti-Igα and anti-50-kD proteins (and bound antibodies) and then eluting the bound antibodies. The affinity-purified antibodies, as well as uncut antiserum, were then used to probe a Western blot of unfractionated LDM. The uncut serum (lane 1) recognized both the 32-kD Igα (arrow B) as well as the 50-kD protein (arrow A) in the LDM. Although the affinity-purified anti-Igα (lane 2) did recognize Igα, it failed to recognize the 50-kD protein present in the LDM. (In some experiments, the affinity-purified anti-Igα antibodies did demonstrate reactivity towards the 50-kD protein.) Importantly, antibodies affinity purified on the 50-kD protein (lane 3) recognized both the 32-kD Igα protein (arrow B) as well as the 50-kD protein (arrow A), indicating an immunological relationship between these two proteins. (Immunoreactivity of the affinity-purified anti-50-kD antibodies against Igα was observed in every experiment.) Illustrated are results representative of three independent experiments.

Mentions:
To this end, we examined the steady state distribution of the Igα subunit of the BCR. As shown in Fig. 5 A, the majority of the 32-kD Igα protein (arrow B) is present in PM-containing FFE fractions with lesser amounts detected in endosome–lysosome and CIIV-containing fractions. The same distribution was also found for the Igβ subunit of the BCR as well as the heavy and light chain subunits of both the huBCR and muBCR (data not shown), suggesting that the subunit composition of the BCRs in these compartments is similar. Surprisingly, the anti-Igα antiserum also recognized a second, 50-kD protein (Fig. 5 A, arrow A) that appears to be selectively enriched in CIIV-containing FFE fractions.

Bottom Line:
We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs.Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative.The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing.

Affiliation:
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06520-8002, USA. jdrake@northnet.org

ABSTRACTB cell receptor (BCR)-mediated antigen processing is a mechanism that allows class II-restricted presentation of specific antigen by B cells at relatively low antigen concentrations. Although BCR-mediated antigen processing and class II peptide loading may occur within one or more endocytic compartments, the functions of these compartments and their relationships to endosomes and lysosomes remain uncertain. In murine B cells, at least one population of class II- containing endocytic vesicles (i.e., CIIV) has been identified and demonstrated to be distinct both physically and functionally from endosomes and lysosomes. We now demonstrate the delivery of BCR-internalized antigen to CIIV within the time frame during which BCR-mediated antigen processing and formation of peptide-class II complexes occurs. Only a fraction of the BCR-internalized antigen was delivered to CIIV, with the majority of internalized antigen being delivered to lysosomes that are largely class II negative. The extensive colocalization of BCR-internalized antigen and newly synthesized class II molecules in CIIV suggests that CIIV may represent a specialized subcellular compartment for BCR-mediated antigen processing. Additionally, we have identified a putative CIIV-marker protein, immunologically related to the Igalpha subunit of the BCR, which further illustrates the unique nature of these endocytic vesicles.